Orientation-Locked DNA Origami for Stable Trapping of Small Proteins in the Nanopore Electro-Osmotic Trap

Nanopores are versatile single-molecule sensors offering a simple label-free readout with great sensitivity. We recently introduced the nanopore electro-osmotic trap (NEOtrap) which can trap and sense single unmodified proteins for long times. The trapping is achieved by the electro-osmotic flow (EOF) generated from a DNA-origami sphere docked onto the pore, but thermal fluctuations of the origami limited the trapping of small proteins. Here, we use site-specific cholesterol functionalization of the origami sphere to firmly link it to the lipid-coated nanopore. We can lock the origami in either a vertical or horizontal orientation which strongly modulates the EOF. The optimized EOF greatly enhances the trapping capacity, yielding reduced noise, reduced measurement heterogeneity, an increased capture rate, and 100-fold extended observation times. We demonstrate the trapping of a variety of single proteins, including small ones down to 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.

F ew techniques have the ability to sense single biomolecules in a label-free manner, and even fewer can do so in solution and at room temperature. The recently developed nanopore electro-osmotic trap (NEOtrap) is such a label-free single-molecule technique that can trap and study proteins one by one. 1,2 As shown in Figure 1a, the NEOtrap consists of a DNA-origami sphere that is docked onto a passivated solid-state nanopore when a positive bias voltage is applied (to the trans side). Once docked, the highly negatively charged DNA-origami sphere generates an electro-osmotic flow (EOF) by which a target protein can be trapped ( Figure  1b). Various protein-specific features, such as protein size and distinct conformations, can be monitored by recording the through-pore ionic current. This electrical readout provides the NEOtrap with a broad temporal bandwidth compared to other single-molecule techniques: 3 big proteins, such as ClpP (340 kDa), in suitable conditions (proper pore size and bias voltage) can be trapped and observed for up to hours with a time resolution of microseconds. 1 However, it appeared challenging to trap small proteins for a long time in the NEOtrap. As shown in Figure 1c, avidin (54 kDa; ∼6 nm in diameter 4 ) exhibits a typical trapping time of only milliseconds. We hypothesized that the short trapping time is likely limited by thermal positional fluctuations of the DNA-origami sphere, allowing the through-pore escape of the protein. This led us to a new NEOtrap design which we report in the current Letter.
Here, we present the "NEOtrap 2.0" with significantly enhanced trapping and sensing performance down to small proteins, which is achieved by two advances: First, we block the through-pore escape pathway by firmly attaching the DNAorigami sphere to the nanopore. Second, guided by numerical simulations, we conceived a new way to tune the magnitude of the EOF in situ, namely by controlling the orientation of the docked origami sphere. We realized this orientation locking experimentally and showed a strong orientation dependence of the EOF in protein trapping experiments. Finally, we demonstrate the enhanced sensing performance of the "vertically" orientation-locked NEOtrap, based on a variety of proteins in a size range down to 13.7 kDa. Remarkably, we find a 100-fold increase of the trapping times, more homogeneous trapping, and significantly reduced noise compared to the previous design.
To improve the trapping capacity of the NEOtrap, we locked the DNA-origami sphere onto the lipid-bilayer-coated nanopore by attaching cholesterol molecules to its surface. We coupled 12 cholesterols to the origami, one at each of the 12 corners of the icosahedral origami structure (see Supporting Note 1 and Figure S1 in the Supporting Information (SI)). Cholesterol molecules are known to insert into lipid bilayers owing to their strong hydrophobic interaction with amphiphilic molecules. Here, they thus act as anchors that firmly attach the sphere onto the nanopore (see Figure 2a). This can be observed in our experiments by comparing the current recordings for cholesterol-functionalized origami versus traces for bare spheres: without functionalization (Figure 2b), application of a negative bias ejected the negatively charged origami, and the open-pore current was recovered when flipping back to positive voltage, shortly thereafter followed by a new origami docking event. By contrast, the cholesterolfunctionalized DNA-origami sphere ( Figure. 2c) was quasipermanently attached to the nanopore upon its first docking, and it stayed firmly docked during repeated voltage inversions, leading to the observed constant current levels in Figure 2c.
We then tested at which voltage the DNA-origami sphere detached from the pore, using voltage ramps from positive to negative bias (see SI Note 2 and Figures S2−S4). As shown in Figure 2d, without cholesterol functionalization, the DNAorigami sphere detached already at small positive voltages (of about +50 mV), where thermal fluctuations were sufficient to overcome the electrophoretic docking potential. By contrast, the cholesterol-functionalized sphere stayed attached even up to considerable negative voltages, before detachment at a voltage between −200 and −800 mV. At these high applied voltages, the lipid bilayer coating got destabilized, causing increased noise. In some cases, the lipid bilayer was even totally peeled off (see SI Figure S5), which suggests that the cholesterols on the origami sphere pulled out lipids from the bilayer during rupture.
Gratifyingly, the cholesterol-functionalized spheres showed significantly reduced current noise. Comparing the standard deviation σ of the current fluctuations, the cholesterolfunctionalized sphere showed typical values of σ = 8 pA, which is close to the open-pore baseline of the same lipidcoated pore (σ = 7pA) and ∼40% less than that for nonfunctionalized spheres (σ = 13 pA, all measured at 100 mV and 10 kHz bandwidth). These values convert to an increase in signal-to-noise ratio (⟨ΔI⟩/σ with ΔI = I open − I dock ) of 32.5 for the cholesterol-functionalized sphere compared to 21.5 for the bare origami sphere. As shown in the power spectrum in Figure 2e, the noise reduction originates predominantly from a low-frequency 1/f-type noise (<1 kHz) which is reduced by ca. an order of magnitude upon cholesterol functionalization. This can be attributed to reduced thermal fluctuations, as 1/f low-frequency noise has been ascribed to mechanical instabilities. 5 Altogether, the cholesterol functionalization strongly reduces the excess noise�almost to the open-pore level.
We found that the orientation of the DNA-origami sphere on top of the nanopore is of great interest. Notably, the "sphere" is only pseudoisotropic, as it is built of many parallel DNA helices (cf. Figure 2a). Accordingly, it can dock onto the pore in various orientations. To estimate the effect of such different origami orientations on the EOF, we first performed finite-element simulations using the COMSOL Multiphysics software. We simulated our experimental conditions using an axis-symmetrical two-dimensional approximation, with an origami sphere mimicked using "DNA rods", void nanochannels, and corresponding parameters. As illustrated in Figure 3a, we considered the two extreme cases of a vertically or horizontally oriented sphere, where the DNA helices are parallel or perpendicular to the pore axis, respectively. The electric field, ion transport, and water movement were fully coupled, as described by the Poisson, Nernst−Planck, and Navier−Stokes equations, respectively 6 (see note 1 in the SI for details). These simulations yielded the EOF fields and the water and ion flows, and Figure 3a shows the substantially different EOF distribution for both sphere orientations (also see Figure S6 in the SI). Clearly, vertical sphere docking causes a much stronger EOF through the nanopore, compared to the horizontal configuration, leading to a water velocity for the vertical docking that is significantly higher than that for the horizontal docking. This can be intuitively understood as a result of a less obstructed EOF in the vertical case. An approximately 2.5 times higher flow rate was found for the    We realized orientation-locked docking of the DNA-origami sphere on the lipid-coated nanopore (Figure 4a) by attaching six cholesterol molecules at specific locations of the sphere, instead of the uniform distribution discussed above. In the "vertical design", cholesterol molecules were attached only at one end of the DNA double helices, whereas the "horizontal design" featured cholesterols at one side plane of the DNA origami spheres (see Figure S1 in SI for details). The two orientations of docking showed distinct current blockades: the vertical orientation generated a deeper relative blockage (17% of the open-pore current) compared to the horizontal counterpart (14.5% of the open-pore current; both measured

Nano Letters pubs.acs.org/NanoLett
Letter on the same pore) (see Figure 4b and SI Figure S7). We attribute this to the nonperfect sphere geometry where the tips of the central helices can fit deeper into the pore for the vertically oriented sphere and thus block more through-pore current than for the horizontally oriented sphere. In the docking experiments, we typically observed that voltage inversions first led to sphere release and baseline recovery a few times, until a final permanent (i.e., voltage-inversionresistant) origami-sphere arrangement was realized with the designed orientation on the nanopore (see for example Figure  4b). The vertical design was observed to need fewer attempts than the horizontal design to achieve the permanent docking arrangement. Next, we tested the protein trapping properties of the vertically and horizontally orientation-locked spheres. Indeed, the two docking orientations led to significantly different trapping observations, as shown in the current traces for avidin trapping in Figure 4c. In line with our simulation results, the vertical docking showed a higher capture rate (7.7 molecules/ s) and a reduced escape rate (11 molecules/s), leading to more frequent and longer trapping events, as compared to those of the horizontal docking. From the scatter plot (Figure 4d), it is clear that the vertical configuration can trap avidin with a welldefined deeper blockade and for a much longer time up to ∼100 ms, as compared to the ∼0.1 ms in the horizontal case. Notably, the same trends are found for other proteins, such as ovalbumin (see SI, Figure S8). As quantified in Figure 4e and 4f, we consistently found that the vertical configuration showed a significantly faster capture rate (by a factor of 2) and a slower escape rate (factor of 270), leading to favorable longer trapping and sensing times. The fact that the vertical configuration gave similar trapping times as those found for spheres without specific orientation locking (i.e., with the uniformly cholesterol-functionalized DNA-origami spheres: 50 ms for avidin at 100 mV) indicates that the vertical orientation is the naturally preferred docking orientation. We suggest that this tendency to orient the origami sphere vertically onto the nanopore is caused by a geometric alignment of the origami's DNA helices with the electric field (consistent with an anisotropic ion mobility found in DNA-origami structures 7 ), while the sphere approaches the nanopore electrophoretically.
The experimental protein trapping results strongly support the notion of an orientation dependence of the EOF, as proposed by the numerical simulations. The increased capture rate and reduced escape rate indicate a larger hydrodynamic trapping potential due to more EOF if the DNA-origami is vertically oriented rather than horizontally. This can be understood by considering the microscopic structure of the DNA-origami sphere where DNA helices are arranged in a honeycomb lattice that defines intermediate nanochannels of 1−2 nm in diameter. In the vertical configuration, these nanochannels are aligned with the main direction of the EOF, which is caused by the positive bias voltage acting on the counter cations that screen the DNA's negative charge. The electrical field drives these accumulated cations upward along the nanochannels, which drags water molecules along to form the EOF. However, in the horizontal configuration, the nanochannels are aligned perpendicularly and thus impede the vertical ion and water flow, adding additional friction. In addition, in the vertical configuration, the DNA-origami structure reached deeper into the pore (as shown by the deeper current blockade in Figure 4b), and thus the electroosmotically active DNA material was exposed to stronger electric fields, causing more hydrodynamic flow. The viscous shear force acting on a trapped protein in this vertical case can be estimated to be on the order of magnitude of a few pN (SI Note 3). Altogether, the vertical orientation locking presents enhanced EOF and improved trapping properties for the NEOtrap.
Lastly, we directly compared the new vertically orientationlocked design introduced here with the original NEOtrap design. Exploring a set of six proteins of varied sizes, we found that the vertically docked spheres very significantly improved the trapping performance of the NEOtrap by prolonging the trapping time, as shown in Figure 5a. For example, trapping events of avidin, ovalbumin, and carbonic anhydrase showed a 10−100 ms trapping time by using the vertically orientationlocked origami spheres, while it was shorter than 1 ms using the previous nonfunctionalized origami design. Consequently, the vertically locked spheres provide more observation time for dynamic processes that occur in many protein systems. 8 Furthermore, owing to the reduced thermal noise by the cholesterol-induced linkage to the pore, the events show an improved signal-to-noise ratio, and we also found a better reproducibility from experiment to experiment due to the better-defined orientation-locked configuration.
To quantify the achieved longer trapping times, we compiled the trapping time histograms and fitted them with an exponential to extract the characteristic trapping time, i.e., the inverse escape rate. For avidin, the cholesterol functionalization resulted in a 98 ± 8-fold increase of the trapping time for various bias voltages (see SI Figure S9). We examined the trapping of six different proteins with a mass ranging from 13.7 to 340 kDa: ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), avidin (54.3 kDa), dCas9 (160 kDa), and ClpP (340 kDa). An exponential dependence of the trapping time on molecular weight was obtained, in agreement with previous results. 1 We consistently observed that the cholesterol origami spheres offered 100-fold longer observation times of unmodified proteins, as summarized in Figure 5b. We attribute this to the cholesterol-induced pore linkage which blocks the through-pore escape of proteins. The observed systematic increase of the trapping time for all proteins by about 2 orders of magnitude converts to an increase of the trapping potential/energy barrier of ∼5k B T (see SI Note 4). The smallest protein that we examined for trapping with the vertically locked sphere was 13.7 kDa (Ribonuclease A), while notably such small proteins could not be trapped without cholesterol functionalization (see SI, Figure S10).
In this Letter, we presented a NEOtrap 2.0 system with a strongly enhanced trapping capacity, including reduced noise, reduced measurement heterogeneity, increased capture rate, 100-fold extended observation time, and last but not least an increased detectable range of protein masses down to 14 kDa. This was achieved by site-specific cholesterol functionalization, which locks the DNA-origami sphere in a defined vertical orientation onto the nanopore. Damping the thermal fluctuations of the sphere thus reduced low-frequency noise, and the tight linkage to the nanopore minimized through-pore escape routes. As shown by simulations and experimental data, the vertically locked sphere showed an enhanced EOF. Stabilization of docking and control of EOF were realized by the cholesterol anchors, which extend the applicable range of the NEOtrap to smaller proteins. The added control obtained with this new NEOtrap significantly improved the reproduci-